PROJECT SUMMARY The Farzan laboratory has developed what appears to be an effective way to prevent HIV-1 infection. This approach combines eCD4-Ig, an exceptionally broad and potent HIV-1 entry inhibitor, with an adeno- associated virus (AAV) delivery system. For more than a year after a one-time intramuscular inoculation with AAV/eCD4-Ig, rhesus macaques were protected against multiple high-dose intravenous challenges with SHIV-AD8 and SIVmac239. AAV vectors have the potential to persist in muscle for a lifetime. This longevity is an important advantage of the AAV system, but also poses risks. Specifically, while it is likely that eCD4-Ig is safe, it has not yet been expressed in large numbers of people or for long periods of time, and there is no easy way to undo AAV administration in case of adverse events. To that end, we seek here to develop a practical means of halting the expression of an AAV transgene. We propose optimizing AAV vectors to be efficiently inactivated upon the administration of lipid nanoparticles (LNPs) containing a modified mRNA that encodes the CRISPR effector protein Cpf1. The optimized system for shutting off AAV transgene expression will then be tested in mice. This work will allow us to include an off-switch in AAV vectors when they are tested in humans. A practical means for turning off eCD4-Ig expression by AAV will improve the safety of an effective approach for treating or preventing HIV-1 infection.